Abstract: Evidence is rapidly accumulating in support of specific and functionally significant sub- cellular mRNA localization in many organisms. Transcript localization shapes many fundamental processes including cell polarity, migration and neuronal activity, and impacts diverse biological processes such as development, memory and learning. Although attention has been focused on localization of a few selected transcripts, recent global analyses indicate that the phenomenon is extremely common. In many cases, different transcripts are observed to have distinct sub-cellular spatial localization patterns. Several prominent examples of specific transcript localization shaping processes such as body axis polarity in Drosophila and synaptic plasticity in neurons suggest the potential for there being direct functional significance of transcript localization, now recognized to be a commonly observed phenomenon. Unfortunately, we have very limited understanding of the protein factors required for achieving specific transcript localization patterns. Moreover, we lack strategies for selectively perturbing transcript spatial distribution in a manner compatible with understanding the associated functional consequences. This proposal addresses these needs by introducing a method permitting regulated targeting of a given transcript to a sub-cellular location, the latter being driven by known or putative localization factors under evaluation. In this approach, transcript localization is entirely experimentally controlled, and is conditional upon either small molecule or peptide signals applied to target cells using high-resolution chemical gradients and post-translational protein localizing modifications, respectively. This broadly applicable method has the potential to advance our basic knowledge of the cellular mechanisms underlying transcript localization, and to probe the associated functional implications at both the cellular and organism levels. Public Health Relevance: Sub-cellular RNA localization into discrete, transcript-dependent patterns is now recognized to be a widespread phenomenon in many cell types and organisms, including humans. In several well-studied cases, transcript localization is required for establishing proper cellular function, and defects in this process contribute to developmental, cognitive and other neurological problems in humans. The proposed research will introduce new methods for understanding and manipulating the mechanisms underlying RNA sub-cellular localization, and has the potential to improve both our understanding and treatment of disease processes associated with RNA localization defects.